Faculty Bibliography

Brain structures derived from the mesencephalon (mes) and rhombomere 1 (r1) modulate distinct motor and sensory modalities. The precise origin and cellular behaviors underpinning the cytoarchitectural organization of the mes and r1, however, are unknown. Using a novel inducible genetic fate mapping approach in mouse, we determined the fate and lineage relationships of mes/r1 cells with fine temporal and spatial resolution. We demonstrate that the mes and r1 are neuromeres that along with the isthmic organizer are partitioned along the anterior-posterior axis by lineage restriction boundaries established sequentially between E8.5 and E9.5. Furthermore, a small group of cells originating from the most posterior mes exhibit anterior intracompartmental expansion and contribute throughout the inferior colliculus. Finally, we also uncovered transient and differential genetic lineages of ventral midbrain dopaminergic and ventral hindbrain serotonergic neuronal precursors with respect to Wnt1 and Gli1 expression

Cystic fibrosis (CF) airway epithelial cells are more susceptible to viral infection due to impairment of the innate host defense pathway of nitric oxide (NO). NO synthase-2 (NOS2) expression is absent, and signal transducer and activator of transcription (STAT) 1 activation is reduced in CF. We hypothesized that the IFN-gamma signaling pathway, which leads to NOS2 gene induction in CF airway epithelial cells, is defective. In contrast to a lack of NOS2 induction, the major histocompatibility complex class 2, an IFN-gamma-regulated delayed-responsive gene, is similarly induced in CF and non-CF airway epithelial (NL) cells, suggesting an NOS2-specific defect in the IFN-gamma signaling pathway. STAT1 and activator protein-1, both required for NOS2 gene expression, interact normally in CF cells. Protein inhibitor of activated STAT1 is not increased in CF cells. IFN-gamma induces NOS2 expression in airway epithelial cells through an autocrine mechanism involving synthesis and secretion of IFN-gamma-inducible mediator(s), which activates STAT1. Here, CF cells secrete IFN-gamma-inducible factor(s), which stimulate NOS2 expression in NL cells, but not in CF cells. In contrast, IFN-gamma-inducible factor(s) similarly inhibit virus in CF and NL cells. Thus autocrine activation of NOS2 is defective in CF cells, but IFN-gamma induction of antiviral host defense is intact.

The last steps of cysteine biosynthesis are catalysed by a bi-enzyme complex composed of serine acetyltransferase (SAT) and cysteine synthase, also called O-acetyl-serine (thiol) lyase (OASTL). SAT is responsible for the production of O-acetyl-serine (OAS) from serine and acetyl-coenzyme A, while OASTL catalyses the formation of cysteine from OAS and hydrogen sulphide. Several distinct nuclear genes for SAT and OASTL enzymes exist in plants. Products of these genes are targeted into at least three cellular compartments: cytosol, chloroplasts, and mitochondria. The SAT and OASTL enzymes are strongly evolutionary conserved, both structurally and functionally. Therefore, isoenzymes from various cellular compartments can be substituted, not only by their plant counterparts from the other cellular compartments but also by their bacterial homologues. During the last decade transgenic plants overproducing SAT, OASTL or both enzymes simultaneously were obtained independently by several research groups. These manipulations led not only to the elevated levels of the respective products, namely OAS and cysteine, but also to increased amounts of glutathione and changes in the levels of other metabolites and enzymatic activities. In several cases, the transgenic plants were also shown to be less susceptible to applied abiotic stresses. In this review, all published and some unpublished results from this laboratory related to heterologous overproduction of SAT and OASTL in transgenic plants are discussed and summarized.

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence

Research carried out in mammalian epithelial cell systems over the past 25 years has delineated pathways and sorting signals involved in polarized delivery of plasma membrane proteins. Recently some progress has been made in the identification of mechanisms underlying this polarized trafficking and in the visualization of trafficking routes in live cells. A promising area of research is the study of trafficking functions of novel polarity genes identified in Drosophila and Caenorhabditis elegans.

Aging and apolipoprotein E (APOE) isoform are among the most consistent risks for the development of Alzheimer's disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by-products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid beta (Abeta) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEepsilon3 or human APOEepsilon4 alleles]. Aging-, gender-, and APOE-genotype-dependent changes were observed for endogenous mouse brain Abeta40 and Abeta42 peptides. Levels of the oxidized lipid F2-isoprostane (F2-isoPs) in the brains of the same animals as those used for the Abeta analyses revealed aging- and gender-dependent changes in APOE KO and in human APOEepsilon4 transgenic KO mice. Human APOEepsilon3 transgenic KO mice did not exhibit aging- or gender-dependent increases in F2-isoPs. In general, the changes in the levels of brain F2-isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Abeta levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging-dependent, APOE-genotype-sensitive rise in murine brain Abeta levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEepsilon3, but not human APOEepsilon4, can apparently prevent the aging-dependent rise in murine brain Abeta levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Abeta/F2-isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2-isoPs can stimulate Abeta generation and aggregation

The trafficking of circulating stem and progenitor cells to areas of tissue damage is poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) mediates homing of stem cells to bone marrow by binding to CXCR4 on circulating cells. SDF-1 and CXCR4 are expressed in complementary patterns during embryonic organogenesis and guide primordial stem cells to sites of rapid vascular expansion. However, the regulation of SDF-1 and its physiological role in peripheral tissue repair remain incompletely understood. Here we show that SDF-1 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of SDF-1 in ischemic tissue in direct proportion to reduced oxygen tension. HIF-1-induced SDF-1 expression increases the adhesion, migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue. Blockade of SDF-1 in ischemic tissue or CXCR4 on circulating cells prevents progenitor cell recruitment to sites of injury. Discrete regions of hypoxia in the bone marrow compartment also show increased SDF-1 expression and progenitor cell tropism. These data show that the recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by hypoxic gradients via HIF-1-induced expression of SDF-1

Pulsed electromagnetic fields (PEMF) have been shown to be clinically beneficial, but their mechanism of action remains unclear. The present study examined the impact of PEMF on angiogenesis, a process critical for successful healing of various tissues. PEMF increased the degree of endothelial cell tubulization (sevenfold) and proliferation (threefold) in vitro. Media from PEMF cultures had a similar stimulatory effect, but heat denaturation ablated this activity. In addition, conditioned media was able to induce proliferative and chemotactic changes in both human umbilical vein endothelial cells and fibroblasts, but had no effect on osteoblasts. Angiogenic protein screening demonstrated a fivefold increase in fibroblast growth factor beta-2 (FGF-2), as well as smaller increases in other angiogenic growth factors (angiopoietin-2, thrombopoietin, and epidermal growth factor). Northern blot analysis demonstrated an increase in FGF-2 transcription, and FGF-2 neutralizing antibody inhibited the effects of PEMF. In vivo, PEMF exposure increased angiogenesis more than twofold. We conclude that PEMF augments angiogenesis primarily by stimulating endothelial release of FGF-2, inducing paracrine and autocrine changes in the surrounding tissue. These findings suggest a potential role for PEMF in therapeutic angiogenesis

Aldose reductase (AR), a member of the aldo-keto reductase family, has been implicated in the development of vascular and neurological complications of diabetes. Recently, we demonstrated that aldose reductase is a component of myocardial ischemic injury and that inhibitors of this enzyme protect rat hearts from ischemia-reperfusion injury. To rigorously test the effect of aldose reductase on myocardial ischemia-reperfusion injury, we used transgenic mice broadly overexpressing human aldose reductase (ARTg) driven by the major histocompatibility complex I promoter. Hearts from these ARTg or littermate mice (WT) (n=6 in each group) were isolated, perfused under normoxic conditions, then subjected to 50 min of severe low flow ischemia followed by 60 min of reperfusion. Creatine kinase (CK) release (a marker of ischemic injury) was measured during reperfusion; left ventricular developed pressure (LVDP), end diastolic pressure (EDP), and ATP were measured throughout the protocol. CK release was significantly greater in ARTg mice compared with the WT mice. LVDP recovery was significantly reduced in ARTg mice compared with the WT mice. Furthermore, ATP content was higher in WT mice compared with ARTg mice during ischemia and reperfusion. Infarct size measured by staining techniques and myocardial damage evaluated histologically were also significantly worse in ARTg mice hearts than in controls. Pharmacological inhibition of aldose reductase significantly reduced ischemic injury and improved functional recovery in ARTg mice. These data strongly support key roles for AR in ischemic injury and impairment of functional and metabolic recovery after ischemia. We propose that interventions targeting AR may provide a novel adjunctive approach to protect ischemic myocardium